Sc 271595

Cells were lysed in IP buffer containing phenylmethylsulfonylfluoride (PMSF), and proteinase inhibitors for 30 min on ice. The cell lysates were centrifuged at 10,000 rpm for 10 min at 4 °C to collect supernatants. Protein concentrations were measured with BCA protein assay kit. Proteins were electrophoresed and transferred to PVDF membranes and incubated with antibody. Finally, the bands were quantified using ImageJ software. The following primary antibodies: P62 (1:1000, Sigma, P0067), LC3 (1:1000, Sigma, L7543), SOD1 (1:2000, Abcam, ab16831), HMGCR (1:200, Santa, sc-271595), FDPS (1:100, Proteintech, 16129-1), RABGGTA (1:100, Proteintech, 14448-1), Rab7 (1:500, Abcam, ab137029), and β-actin (1:1000, Proteintech, 60008-1).

Proteins were extracted using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, 23225, Waltham, MA, USA). Equal amount of proteins (40 μg) were separated by 10 or 12% SDS-PAGE and then transferred to PVDF membranes. After blocking by 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C, and then washed three times (10 min/time), subsequently with secondary antibodies for 1 h at room temperature. Finally, the bands on the membranes were scanned with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA), and quantities analysis was performed with ImageJ.
The primary antibodies: P62 (1:1000, Sigma, P0067), LC3 (1:1000, Sigma, L7543), SOD1 (1:2000, Abcam, ab16831), HMGCR (1:200, Santa, sc-271595), FDPS (1:100, Proteintech, 16129-1), RABGGTA (1:100, Proteintech, 14448-1), Rab7 (1:500, Abcam, ab137029), and β-actin (1:1000, Proteintech, 60008-1).